Manufacturing of biopharmaceuticals depends on cell cultures that secrete protein products into surrounding media. Typically, this conditioned media containing the desired product is used for downstream processing, while a new batch of fresh media is supplied to the cells. Naturally, increasing cell density in the bioreactor can make the process more productive. However, the lifespan of super-dense cultures is much shorter than lower density cultures because continuous proliferation can reduce available attachment surfaces until cell layers detach from solid support. At this stage, the bioreactor has to be recycled: washed, sterilized and re-seeded.
Cell growth can lead to increased product yield until a maximum is reached; then the cycle is repeated. During this cycle the period of maximum cell density and maximum bioreactor efficiency can be relatively short. The length of this period is determined to a large extent by the proliferation rate of producer cells. At the beginning of the cycle, when the seeding density is relatively low, it is advantageous to INCREASE proliferation rate to achieve high cell density faster. On the other hand, it would be favorable to REDUCE the rate of proliferation when the maximal density is achieved in order to preserve cell population at its most productive state. Besides increasing the bioreactor cycle at its production peak, reduced rates of proliferation can channel cell energy from proliferation to protein production, further increasing yields.
Current approaches to increasing the useful time of bioreactor cycle concentrate on media adjustments at or close to the peak of production. The most common method is reduction in serum content of the bioreactor media. While effective in preventing further cell division, this approach can interfere with protein synthesis, thus reducing beneficial effects of decreased cell growth.
Recombinant DNA technology has opened new avenues for the production of useful therapeutic proteins, such as hormones, growth factors, and interferons, in commercial quantities. To economically produce therapeutic proteins at commercial scale, while controlling product quality requires three general steps. First, an effective strategy for maximizing recombinant gene expression, next, a sufficient fermentation process, finally, robust protein recovery and purification processes must be instated.
Elaborate methods of vector construction and cell culture methods are required for production of biopharmaceuticals from mammalian cells. Promoters such as immediate early cytomegalovirus promoter (CMV) can mediate very strong interactions with the transcriptional machinery in most mammalian cellular systems (F. Weber, J. de Villiers, W. Schaffner, Cell 36, 983-92 (1984). Based on this attribute, CMV promoter is frequently used in mammalian expression vectors. High concentrations of protein (mg/ml) are generated using these constructs. The limitation in production of these biopharmaceuticals is generally related to the capacity of the cells to synthesize and secrete the protein product.
Developments in bioprocess engineering of mammalian cells have generally relied on manipulation of culture media components to reduce cell proliferation upon achieving a high density of cells. When the bioreactor is initially seeded with protein-producing cells the efficiency of the process is relatively low because of the low cell density. At this stage cell growth in the bioreactor is the major concern; however, when the cell density reaches its maximum, cell growth becomes detrimental to the system, because cells require additional space and nutrients. Decreasing serum in the media is the most common method of blocking cell proliferation, however, in many cases the core effect of these modifications is reduction of energy level, which is detrimental to the protein synthesis and thus to overall production capacity of the bioreactor.
It would be useful to have a technology that could prevent cell proliferation without affecting protein synthesis to result in increased yields of synthesized bioproducts. An additional benefit of such technology would be diversion of energy, otherwise spent on reproduction, to sustain/increase protein synthesis.
The invention relates to protein synthesis in general, and increased protein synthetic productivity of cells in particular. By switching the cells from a replicative to a productive state (RP switch), protein biosynthesis can be extended. The productive state is a pseudo-senescent state. This pseudo-senescent state can be induced by transforming the cells with a vector expressing a cell cycle inhibitor. Expression of the cell cycle inhibitor within the cell, because it does not cause cell death, allows for cells to be maintained in culture for longer periods (cell numbers within a culture do not increase and overgrow the growth container). The invention allows for controlled enhanced protein biosynthetic productivity of cell lines for commercial and research purposes.
According to one aspect, the invention is a method of increasing yield of a protein from a cell culture, preferably a eukaryotic cell culture, more preferably a mammalian cell culture, by causing a pseudo-senescent state in one or more cells in the cell culture; and collecting a protein fraction from the cell culture.
According to another aspect, the invention is a method of increasing yield of a protein from a eukaryotic cell culture, by contacting the cell culture with an expression vector which comprises an inducible transcription regulation element comprising a tetracycline operator element, and collecting a protein fraction from the cell culture.
According to yet another aspect, the invention is a transcriptional regulatory element which includes a minimal promoter comprising a TATA sequence, two phased tetracycline operators downstream from the TATA sequence, and two phased tetracycline operators upstream of the TATA sequence.
According to still yet another aspect, the invention is an expression vector including a minimal promoter comprising a TATA sequence, two phased tetracycline operators downstream from the TATA sequence, and two phased tetracycline operators upstream of the TATA sequence.
Abbreviations and Definitions
The following abbreviations are used in this disclosure:
CDK, cyclin-dependent kinase;
CKI, cyclin-dependent kinase inhibitors;
CMV, cytomegalovirus;
DNA, deoxyribonucleic acid;
env, the retrovirus gene encoding the envelope proteins in the membrane of the vital particle;
gag, the retrovirus gene encoding the core proteins of the viral particle;
G418, geneticin (GIBCO, Inc);
kb, kilobases of nucleic acid;
LTR, long terminal repeat;
MoMLV (or MuLV), Moloney strain murine leukemia virus;
mRNA, messenger RNA;
neo, neomycin phosphotransferase;
pA, polyadenylation signal;
pol, the retrovirus gene encoding the viral reverse transcriptase;
RNA, ribonucleic acid;
RP, replicative to productive;
PSI, the packaging nucleotide sequence for murine retroviruses;
tTA, Tc-controlled transactivator;
Tet, teracycline;
TetO, tetracycline operon;
TetR, tetracycline repressor;
TFIID, transcription factor IID; and
T-Rex, tetracycline responsive plasmid vector.
xe2x80x9ccell cyclexe2x80x9d means the biochemical process by which mammalian cells duplicate themselves.
xe2x80x9ccyclin-dependent kinasexe2x80x9d is a family of enzymes that trigger progression through the cell cycle.
xe2x80x9cCDK inhibitorsxe2x80x9d are proteins produced naturally by cells to block progression through the cell cycle.
xe2x80x9cdefectivexe2x80x9d, means genetically-deficient in nucleotide sequences required to produce infectious viral particles.
xe2x80x9cdefective retroviral vectorxe2x80x9d, means a retroviral vector containing an incomplete RNA genome capable of infecting a host cell, but incapable of producing a viral infection (i.e., with progeny virus) in that cell which could subsequently infect another cell.
xe2x80x9cdoxycylinexe2x80x9d is a water-soluble tetracycline analog suitable for mammalian cell cultures.
xe2x80x9cecotropic receptorxe2x80x9d is a protein expressed on the membrane of rodent cells that contains the binding site for mouse leukemia viral particles.
xe2x80x9cG418xe2x80x9d is a water-soluble form of neomycin suitable for mammalian cell cultures.
xe2x80x9cpackagedxe2x80x9d, means assembling the recombinant murine retrovirus genome into an infectious retroviral vector by surrounding the recombinant retroviral RNA with the gag and pol proteins to form a core particle and encapsulating the core particle in a membrane containing the env protein.
xe2x80x9cpackaging cellxe2x80x9d, means a cell containing a proviral genome of a first defective retroviral vector that encodes viral proteins sufficient to assemble a second defective retroviral vector into an infectious retroviral vector virion.
xe2x80x9cpromoterxe2x80x9d is a region of DNA where transcription is initiated.
xe2x80x9cproviral genomexe2x80x9d, means a defective retroviral vector nucleic acid integrated in the DNA of a host cell.
xe2x80x9cpseudo-senescentxe2x80x9d cells are cells forced to express a senescent phenotype by manipulation. Pseudo-senescent cells do not include aged cells that have naturally senesced. Cells in a forced or pseudo-senescent state may have prolonged cell culture lifetimes (S. Goldstein, D. P. Singal, Exp Cell Res 88, 359-64 (1974); A. J. Brenner, M. R. Stampfer; and C. M. Aldaz, Oncogene 17, 199-205 (1998)), are resistant to apoptosis (B. D. Chang, et al., Proc Natl Acad Sci USA 97, 4291-6 (2000); and D. Javelaud, J. Wietzerbin, O. Delattre, F. Besancon, Oncogene 19, 61-8 (2000) and increase their protein synthetic capacity several fold (B. D. Chang, et al., Proc Natl Acad Sci USA 97, 4291-6 (2000); V. J. Cristofalo, D. Kritchevsky, Prog Immunobiol Stand 3, 99-105 (1969); and G. H. Stein, L. F. Drullinger, A. Soulard, V. Dulic, Mol Cell Biol 19, 2109-17 (1999).
xe2x80x9cretroviral vectorxe2x80x9d means a genetically-engineered recombinant retrovirus containing a gene of interest, capable of infecting a mammalian cell wherein the gene of interest can become integrated into the genome of the mammalian cell in a manner that promotes the expression of the gene of interest.
xe2x80x9cretrovirusxe2x80x9d, means an infectious RNA virus having an RNA genome that is converted to DNA and integrated into the genome of the host cell.
xe2x80x9cselectingxe2x80x9d, means cloning (e.g., by limiting dilution), killing undesirable cells (e.g., with drugs or toxins), or mechanical (e.g., by fluorescence activated cell sorting) or physical methods (e.g., by microscopic micropipetting) for collecting individual cells with desirable properties.
xe2x80x9csense orientationxe2x80x9d means the direction of DNA sequence in a vector to produce a certain peptide or protein.
xe2x80x9ctetracyclinexe2x80x9d is a naturally occurring antibiotic that is active against bacteria by binding to ribosomes and preventing protein synthesis.
xe2x80x9ctetracycline repressorxe2x80x9d is a helix-turn-helix protein that forms homodimeric complex that binds tightly to the tetracycline operator.
xe2x80x9ctetracycline operatorxe2x80x9d is a 15 bp palindromic sequence (TCCCTATCAGGGAGA; SEQ ID NO: 15) that is bound by tetracycline repressor.
xe2x80x9cTFIIDxe2x80x9d is a core protein necessary for transcription of RNA from DNA at a promoter site.
xe2x80x9ctransactivatorxe2x80x9d means a protein that binds to regulatory regions of DNA and enhances the expression of its associated gene.
xe2x80x9ctranscriptionxe2x80x9d means the process by which the sequence of DNA is used as a template to produce a corresponding piece of RNA.
xe2x80x9cvectorxe2x80x9d means an entity of DNA constructed to introduce and express a set of genes in cells.
xe2x80x9cviral particlesxe2x80x9d is used synonymously with xe2x80x9cvirionsxe2x80x9d to mean an infectious virus having a ribonucleoprotein core particle surrounded by a membrane containing envelope protein.